Ultrastructural and biochemical changes in the liver of monocrotaline intoxicated chickens

TOXICOLOGY

AND

APPLIED

Ultrastructural

PHARhiACOLQGY

16, 800-806

(1970)

and Biochemical Changes in the Monocrotaline Intoxicated Chickens

Liver

of

J. R. ALLEN, L. A. CARSTENS, AND D. H. NORBACK Department of Pathology and Primate Research Center, University of Wisconsin, Madison, Wisconsin 53706 Received June 4,1969

Ultrastructural and BiochemicalChangesin the Liver of Monocrotaline Intoxicated Chickens.ALLEN, J. R., CARSTENS, L. A., and NORBACK, D. H. (1970).Toxicol. Appl. Pharmacol. 16,80&806.Tenweeksafter a single,20-mg oral doseof monocrotalineto chickens,their livers weresmalland irregular in outline and exhibited disruption of the hepatic lobules.There wasalsoa reduction in liver nitrogen, RNA, and DNA content. Over 75% of the surviving hepatocyteswerelarge clear cellsthat containeda sparsepopulation of cytoplasmic organellesin an abundant free matrix and appearedto be functionally quiescent.A smallpopulation of metabolically active hepatocytesformed an irregularnetwork throughout the liver parenchyma.Hepatic failure in theseanimalsresultedfrom a decreasein parenchymalcell population and an alteration in functional capacity of a large percentageof the surviving hepatocytes. Monocrotaline,’ a toxic pyrrolizidine alkaloid, is present in the seedand vegetation of the legume Crotalaria spectabilis. In numerous instances, portions of this plant have contaminated the diet of man and lower animals (Sanders et al., 1936; Gibbons et al., 1950; Bras et al., 1954). Minute quantities of monocrotaline are capable of producing hepatic hemorrhage and necrosis (Harris et al., 1942; Simpson et al., 1963), occlusion of the smaller hepatic vessels(Stuart and Bras, 1956; Berry and Bras, 1957), nodular hyperplasia of the liver (Schoental and Head, 1955) and cirrhosis (Bras et al., 1954; Allen et al., 1963). Previous reports from this (Allen et al., 1960, 1967)as well as other laboratories (Schoental and Head, 1955; Simpson et al., 1963)have elucidated someof the histopathologic changes in monocrotaline-intoxicated animals. However, data correlating the biochemical changeswith histologic and ultrastructural features in the liver of animals following monocrotaline intoxication are lacking. METHODS One hundred 14-day-old New Hampshire-White Leghorn chickens were placed in groups of 75 and 25. Twenty milligrams of monocrotaline suspendedin water was given to each of 75 chickens by gastric intubation. An equal volume of water was administered to the 25 control chickens. All the surviving chickens were sacrificed 10 weeks after the administration of monocrotaline. 1S.B. Penick& Co., 100ChurchStreet,NewYork, NewYork. 800

HEPATOTOXIC

EFFECTS

801

OF MONOCROTALINE

During the course of the experiment, the chickens were given food and water ad libitum. At the termination of the experiment, 5 ml of blood was obtained from the wing vein, and the total serum protein level (Gornall et al., 1949) and serum electrophoretic pattern (Williams et al., 1955) were determined. One hour prior to sacrifice, 5 randomly selected control and 5 treated chickens were given intravenously 10 $Zi of thymidine-3H (16.5 Ci/mmole)2 per 100 g of body weight. The chickens were killed by severing the cervical spinal cord. The abdominal cavity was opened, and the liver was excised and weighed. Small portions of the liver were processed for electron microscopy according to previously reported procedures (Allen et al., 1967). Additional portions ,of the liver were processed for light microscopy, and sections were stained with hematoxylin and eosin and with Masson trichrome stain. Portions of the liver from the chickens receiving thymidine-3H were homogenized with buffer, pH 7.5, for biochemical determinations. Quantities of RNA and DNA were estimated according to the procedure reported by Munro and Fleck (I 966). Theconcentrations of nitrogen in the homogenates were determined by the micro-Kjeldahl procedure (Hawk eial., 1947). A 1.0-ml aliquot of the DNA extract was added to a scintillation fluid and counted in a scintillation spectrometer.3 Counting rates were corrected for quenching by the use of the automatic external standard and for background. All biochemical values were expressed as mean plus or minus standard deviation. Student t distribution was used to evaluate the difference between means of the treated and control groups. The level of significance chosen for all determinations was P s. 0.05. RESULTS

Throughout the course of the experiment the monocrotaline-intoxicated chickens ate well; however, the food consumption was lessthan that of the controls. Anorexia was observed only in those chickens that died during the acute phaseof the intoxication. Fifty-seven of the 75 monocrotaline-treated and all the control chickens survived the experiment. The average survival time of the chickens that died was 8 days (range l-14 days). Gross lesions included extensive subcutaneous edema, ascites, and pulmonary edema, and the livers were large, hemorrhagic, and friable. At the time the chickens were sacrificed, the controls averaged 794 (*40.0) g while the monocrotaline-treated chickens averaged 259 (120.0) g. Forty-five of the surviving 57 treated chickens had from 15-50 ml of ascitic fluid in the abdominal cavity. There wasextensive generalized subcutaneousand pulmonary edema.The livers were firm and small (3.1 g 1;s.16.6 g), and their irregular surfaceswere covered with a gelatinous fluid. The livers from monocrotaline-treated chickens comprised 1.2 % of the body weight. while the control livers averaged 2.1 %. The total serum proteins of the treated and control chickens averaged 2.6 and 3.9 g per 100 ml of serum, respectively. Only 32 % of the serum protein was albumin in the monocrotaline-treated chickens, while in the controls 48 “/, of the protein was albumin. The protein content of the ascitic fluid averaged 2.4 g per 100ml, and the albumin : globulin ratio was similar to that of the blood serum. 2 New England Nuclear Corporation, Boston, Massachusetts. 3 Packard TriCarb scintillation spectrometer, Packard Instrument Illinois.

Co., Inc., Downers

Grove,

802

ALLEN,

CARSTENS,

AND

NORBACK

Light microscopy. Regardless of when the monocrotaline-treated chickens died, the major microscopic alterations were located in the liver. In those chickens that died from acute monocrotaline intoxication, extensive hemorrhage and necrosis caused disruption of the hepatic architecture. The livers of the chickens surviving throughout the experiment had a thick gelatinous capsular covering. With Masson trichrome stain, large portions of this pseudo-capsule assumed a homogeneous blue tinctorial quality characteristic of collagen fibers. Scattered acidophilic fibers near the center of the coagulum resembled fibrin. Extending from the thickened capsule, numerous connective tissue invaginations divided the parenchyma into incomplete segments. The internal morphologic features characteristic of the normal liver (Fig. 1) were disrupted. Centrolobular veins and portal triads were difficult to identify. Approximately 75 % of the hepatic cells in the affected livers were large and pale (Fig. 2). The nuclei were small, pyknotic, and centrally located. Large groups of these cells were arranged in sheets or in well-circumscribed foci throughout the liver. Mitotic figures were not apparent in these cells. Small, deeply stained hepatocytes, many of which were undergoing mitoses, formed an irregular network around groups of the larger cells (Fig. 3). It was estimated that the latter cells comprised 15-20 % of the liver mass. Bile duct proliferation was also evident. These ductal cells were small, cuboidal, and arranged in clusters. Other biliary duct cells were large and surrounded a central lumen. Electron microscopy. Four distinct types of hepatocytes were apparent in the livers of the monocrotaline-intoxicated chickens. Isolated hepatocytes were morphologically similar to those present in the livers of the control chickens (Fig. 4). The ultrastructural features of the normal chicken hepatocytes were similar to those previously described (Allen and Carstens, 1966). The predominant cell type consisted of large electron lucent hepatocytes (Fig. 5). The large lucent areas contained glycogen granules, numerous microvesicles, and large vesicles. The Golgi complex was comprised of a few short lamellar cisternae surrounded by small round vesicles. Rough-surfaced cisternae, mitochondria, lysosomes, and microbodies were also widely distributed throughout the electron lucent areas. The diameter of the third type of cell was approximately one-half that of the electron lucent hepatocyte previously described (Fig. 6). The cytoplasmic organelles were more abundant and the matrix less apparent. The granular endoplasmic reticulum consisted of dilated cisternae filled with fine granular to amorphous material. The smooth endoplasmic reticulum was sparse. Distinct multiple Golgi complexes composed of numerous vesicles and cisternae filled with electron dense material were readily apparent. Mitochondria, lysosomes, microbodies, and nuclei were similar in appearance and frequency to those of the control hepatocytes. The fourth type of hepatocyte had undergone moderate to extensive degenerative changes. Many of these cells were shrunken and electron dense. Large vacuoles, myelin figures, and cytosegresomes were prominent in their cytoplasm. Numerous lysosomes containing cell debris, vacuoles, and dense granules were apparent. The relatively long, flattened lamellae of the rough endoplasmic reticulum were distributed throughout the cytoplasm. There were also widely dispersed slitlike clefts between the cytoplasmic organelles. The electron dense nuclei were shrunken and irregular in outline. The more severely affected cells were extremely electron dense and their various organelles only

HEPATOTOXIC

EFFECTS

OF MONOCROTALINE

803

FIG. 1. The liver lobule of a control chicken depicts a central vein surrounded by numerous uniform hepatocytes. Slender Kupffer cells are obvious in the sinusoidal spaces. Hematoxylin and eosin stain; /120. FIG. 3. Large clear hepatocytes were the predominant cell type in the livers of chickensgiven monocrotaline. Note the lacy appearance of the cytoplasm and the pyknotic nuclei. Hematoxylin and eosin stain; x 150. FIG. 3. Foci of small deeply stained hepatocytes were distributed throughout the livers of the monocrotaline-treated chickens. Hematoxylin and eosin stain; x 130. FIG. 4. A hepatocyte from a control chicken liver. Observe the numerous mitochondria, short segments of rough endoplasmic reticulum, and free ribosomes. Chromatin granules were abundant along the inner surface of the nuclear envelope and clumped throughout the nucleoplasm. Uranyl acetate stain; r11,100.

804

ALLEN,

CARSTENS,

AND

NORBACK

FIG. 5. The large electron lucent hepatic cells of monocrotaline-treated chickens contained abundant organelle-free cytoplasmic matrix (nzx), vacuoles (o), and glycogen (arrow). Note the sparseness of rough endoplasmic reticulum (er). Uranyl acetate and lead citrate stain; x6800. FIG. 6. The smaller, more electron dense hepatocytes of chickens given monocrotaline contained abundant cytoplasmic organelles. Note the prominent vesicular ribosome-associated endoplasmic reticulum (er). Numerous free ribosomes were apparent throughout the cytoplasmic matrix. Uranyl acetate stain; x7100.

TABLE HEPATIC CHANGES IN THE CHICKEN

1 AFTER

MONOCROTALINE

ADMINISTRATION

Variables DNA” (mg/lOO mg liver) RNAb (mg/lOO mg liver) Nitrogen (mg/lOO mg liver) DNA/nitrogen RNA/nitrogen Incorporation of thymidine-3H (dpm/O. 1 mg DNA) Liver weight (g) Relative liver weight (g/100 g body weight)

Treated chickens 0.11 0.33 2.03 0.05 0.16 7190

zt i + It * rt

0.03’ 0.09 0.24 0.01 0.03 4900

3.10 f 0.70 1.20 It 0.20

Control chickens 0.40 0.70 3.07 0.13 0.23 820

It 0.05 It 0.09 * 0.29 z!z0.01 zt 0.01 f 279

16.6 * 1.20 2.09 i 0.30

’ Deoxyribonucleic acid. b Ribonucleic acid. ’ The data for experimental and control groups represent the mean values and standard deviations based on 5 randomly selected animals. In all instances the values for treated chickens were found to be significantly different from the controls (P < 0.05).

HEPATOTOXIC

EFFECTS

OF MONOCROTALINE

805

vaguely discernible. Large segments of these cells had become detached from the cell surface and were free in the extracellular space. In addition to cells representative of the four previously described types, hepatocytes with ultrastructural features characteristic of more than one type were observed. Biochemistry. The biochemical data are presented in Table 1. Concentrations of nitrogen and RNA per wet weight of liver in the tissues of the monocrotaline-treated chickens were considerably less than in the control tissues. The DNA concentrations were reduced approximately by a factor of 4 in the treated animals. Although considerable variation existed in the incorporation of thymidine-3H by the experimental hepatocytes (range 2500-19,000 dpm/O.l mg DNA), the incorporation of the isotope for each experimental chicken exceeded that of any control. Statistically significant (P i 0.05) differences were found in each parameter listed in Table I. DISCUSSION Distinct morphological and biochemical changes occurred in the livers of monocrotaline-intoxicated chickens. During the acute phase of intoxication there was extensive hemorrhage and necrosis. Similar, but less severe, hepatic changes occurred in the livers of chickens that survived. The necrotic tissue and extravasated blood cells gradually decreased (Allen er al., 1960). However, the residual effects of monocrotaline on the surviving hepatocytes facilitated continuous focal hemorrhage and necrosis of the liver (Schoental and Magee, 1959). The large lucent hepatocytes which formed the greater percentage of the surviving cells appeared to be functionally quiescent. These cells were mitotically inactive and contained a sparse population of widely dispersed cytoplasmic organelles. The Golgi complexes were small and devoid of electron dense material within the cisternae. The major functional role of the liver was likely assumed by the small, more dense hepatocytes. These cells contained large, active Golgi complexes and abundant granular endoplasmic reticulum. The high frequency of mitotic figures and the uptake of thymidine-3H indicated the presence of a population of regenerating, metabolically active cells. The biochemical data from the affected livers augmented the light and electron microscopic observations regarding the functional status of the liver. The decrease in nitrogen, RNA, and DNA was related to the dilution factor afforded by the large population of swollen, morphologically altered hepatocytes. Focal necrosis may also have contributed to the reduction of protein and nucleic acid levels in the livers of treated chickens. The data presented in Table 1 indicated that the DNA content of the livers was reduced to a greater extent than other components. The small mitotically active hepatocytes. which were the second most abundant cell type in the affected livers, probably had not attained the degree of polyploidy characteristic of mature liver cells. These small hepatocytes were similar morphologically to the hepatocytes of younger animals which were capable of active division and whose DNA level has been reported to be closer to that of the diploid nuclei (Wannemacher et al., 1968). The microscopic and biochemical changes observed in the livers of these experimental chickens were indicative of hydropic degeneration and suggested that one effect of monocrotaline intoxication included altered membrane permeability. However. the direct action of the monocrotaline was likely several steps removed from this effect.

806

ALLEN, CARSTENS, AND NORBACK ACKNOWLEDGMENT

This investigationwassupportedin part by grantsHE-10941andFR-0167from the National Institutes of Health. REFERENCES ALLEN, J. R., and CARSTENS, L. A. (1966). Electron microscopicalterations in the liver of chickensfed toxic fat. Lab. Invest. 15, 970-979. ALLEN, J. R., CARSTENS, L. A., and OLSON,B. E. (1967).Veno-occlusivediseasein Macaca

speciosamonkeys.Am. J. Pathol. 50,653-667. ALLEN, J. R., CHILDS, G. R., and CRAVENS, W. W. (1960). Crotalaria spectabilistoxicity in chickens.Proc. Sot. Exptl. Biol. Med. 104, 434-436. ALLEN, J. R., LALICH,J. J., and SCHMITTLE, S. C. (1963). Crotalaria spectabilis-induced cirrhosisin turkeys. Lab. Invest. 12, 512-517. BERRY,D. M., and BRAS,G. (1957). Venous occlusioncf the liver in crotalaria and senecio

poisoning.N, Am. Vet. 38, 323-326. BRAS,G., JELLIFFE, D. B., and STUART,K. L. (1954). Veno-occlusivediseaseof liver with nonportal type of cirrhosisoccurring in Jamaica.Arch. Pathol. 57, 285-300. GIBBONS, W. J., HOAKSON, J. F., WIGGINS,A. M., and SCHMITZ,M. B. (1950).Cirrhosisof the liver in horses.N. Am. Vet. 31, 229-233. GORNALL,A. G., BARDAWILL,C. J., and DAVID, M. M. (1949). Determination of serum proteins by meansof the biuret reaction. J. Biol. Chem.177, 751-766. HARRIS,P. N., ANDERSON, R. C., and CHEN,K. K. (1942).The action of monocrotalineand retronecine.J. Pharmacol.Exptl. Therap.75, 78-82. HAWK, P. B., OSER,B. L., and SUMMERSON, W. H. (1947).Practical PhysiologicalChemistry, 12th ed., pp. 820-822.Blakiston, Philadelphia. MUNRO,H. N., and FLECK,A. (1966).The determination of nucleic acids.Methods ofBiochemicalAnalysis (D. Glick, ed.), Vol. 14, pp. 113-176.Wiley (Interscience)New York. SANDERS, D. A., SHEALY,A. L., and EMMEL,M. W. (1936). The pathology of Crotalaria spectabilisroth poisoningin cattle. J. Am. Vet. Med. Assoc.89, 150-162. SCHOENTAL, R., and HEAD,M. A. (1955).Pathologicalchangesin rats asa result of treatment with monocrotaline.&it. J. Cancer9, 229-237. SCHOENTAL, R., and MAGEE,P. N. (1959).Further observationson the subacuteand chronic liver changesin rats after a singledoseof various pyrrolizidine Senecioalkaloids.J. Pathol. Bacterial. 78, 471-482. SIMPSON, C. F., WALDRUP,P. W., and HARMS,R. H. (1963). Pathologic changesassociated with feedingvarious levelsof Crotalaria spectabilis.J. Am. Vet. Med. Assoc.142, 264-271. STUART,K. L., and BRAS,G. (1956). Veno-occlusivediseaseof the liver in Barbados. West Indian Med. J. 5, 33-36. WANNEMACHER, R. W., JR.,MURAMATSU,K., and COOPER, W. K. (1968).Sizedistribution and counting of liver nuclei by an electronic particle counter. Proc. Sot. Exptl. Biol. Med. 129, 899-901.

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